Lab 14 Goals and Objectives:
Exercise 69: Staphylococci Identification
Read and record results
Exercise 70: Streptococci & Enterococci Identification
Read and record results
Repeat Gram stains if necessary for sketch for report from old smears or
plates (note: plates are not “fresh” so Gram results may be variable, but
size, shape and arrangement will be fine).
If you have identified a particular assay you need to repeat to be able to
solve your unknown, the media may be bought for ten points off your
unknown report grade (per assay). Catalase (hydrogen peroxide) and
Gram stain (staining reagents) assays are free.
Be sure you completely understand the lab report format: ask
questions now!
Mannitol Salt Agar
Inoculation method: surface streak with loop
Contains: 7.5% NaCl, mannitol, Phenol red pH indicator: alkaline pH =
red/pink, acidic pH = yellow
Selective and differential medium: selects for growth of organisms salt
tolerant to 7.5% (usually Staphylococci). Of those that grow,
differentiates organisms that have the ability to ferment mannitol to
acid.
Results: Growth = salt tolerant to 7.5%
Yellow = positive for mannitol fermentation to acid
Pink = negative for mannitol fermentation to acid
No growth = negative for salt tolerance to 7.5%, inconclusive
for mannitol fermentation
Growth = colonies
don’t count a heavy
streak only as growth!
Blood Agar
Inoculation method: surface streak and stab with loop
Contains: BHIA, sheep blood
Discriminates organisms that have the ability to hemolyse red blood cells
completely through production of hemolysins (streptolysins or alphatoxin) or partially through ability to degrade hemoglobin pigment into
green products (biliverdin)
Results:
Complete clearing of RBCs = Beta-hemolysis, positive for production
of hemolysins
Partial clearing and greening of blood = Alpha-hemolysis, positive for
degradation of hemoglobin pigment into biliverdin
No clearing with or without rusting = Gamma-hemolysis, negative for
hemolysis
Gamma
Alpha
Beta
Partial clearing and
greening of blood =
Alpha-hemolysis,
positive for
degradation of
hemoglobin
pigment into
biliverdin
Complete clearing of
RBCs =
Beta-hemolysis,
positive for
production of
hemolysins
No clearing with or
without rusting =
Gamma-hemolysis,
negative for hemolysis
Rabbit Serum: Coagulase Test
Inoculation method: loop transfer
Contains: rabbit serum (fibrinogen)
Discriminates organisms that can produce coagulase to catalyze the
conversion of soluble fibrinogen into insoluble fibrin (clot)
Results: Solid = positive for fibrin formation, positive for coagulase
production
Liquid = negative for coagulase production
+
_
Blood Agar
Inoculation method: surface streak and stab with loop
Contains: BHIA, sheep blood
Discriminates organisms that have the ability to hemolyse red blood cells
completely through production of hemolysins (streptolysins or alphatoxin) or partially through ability to degrade hemoglobin pigment into
green products (biliverdin)
Results:
Complete clearing of RBCs = Beta-hemolysis, positive for production
of hemolysins
Partial clearing and greening of blood = Alpha-hemolysis, positive for
degradation of hemoglobin pigment into biliverdin
No clearing with or without rusting = Gamma-hemolysis, negative for
hemolysis
Gamma
Alpha
Beta
Bacitracin Susceptibility or Resistance
Susceptible = organism killed
bacitracin: zone of no growth
around disk
Resistant = organism growth not
affected by bacitracin: organism
grows around and under disk
Bile Esculin Hydrolysis Slant
Inoculation method: surface streak with loop
Contains: esculin, ferric citrate (reacts with hydrolyzed esculin
(esculetin) to produce black precipitate)
Discriminates organisms that can hydrolyze esculin into esculetin and
dextrose
Results: Black = positive for esculin hydrolysis
Colorless = negative for esculin hydrolysis
_
+
Salt Tolerance Broth
Inoculation method: loop transfer
Contains: 6.5% NaCl, Bromcresol purple pH indicator: alkaline pH =
purple, acidic pH = yellow (any growth should cause acid
accumulation)
Discriminates organisms that display salt tolerance to 6.5%
Results: Yellow = positive for salt tolerance to 6.5%
Purple but turbid = positive for salt tolerance to 6.5%
Purple = negative for salt tolerance to 6.5%
_
+
12 Possible Unknowns
Gram Negative
Gram Positive
Bacillus subtilis
Catalase +
Gelatinase -
Gelatinase +
Lactose +
Catalase -
Pseudomonas aeruginosa
Gelatinase +
Gelatinase -
Lactose -
Gram Positive
Gelatinase +
Gelatinase -
Catalase +
Staph assays: Choose one
-Mannitol fermentation
-Hemolysis
-Coagulase
Catalase -
Strep/Entero assays:
Choose one
-Hemolysis
-Bacitracin sensitivity
-Bile esculin hydrolysis
-Salt tolerance to 6.5%
Bacillus
subtilis
EXAMPLE!!!!
Gram Positive
Gelatinase +
Gelatinase -
Catalase +
Coagulase +
Bacillus
subtilis
Staphylococcus
aureus
Coagulase -
Staphylococcus
epidermidis
Catalase -
Esculin +
Enterococcus
faecalis
Esculin -
Streptococcus
pyogenes
Lab 14 Goals and Objectives:
Exercise 69: Staphylococci Identification
Read and record results
Exercise 70: Streptococci & Enterococci Identification
Read and record results
Repeat Gram stains if necessary for sketch for report from old smears or
plates (note: plates are not “fresh” so Gram results may be variable, but
size, shape and arrangement will be fine).
If you have identified a particular assay you need to repeat to be able to
solve your unknown, the media may be bought for ten points off your
unknown report grade (per assay). Catalase (hydrogen peroxide) and
Gram stain (staining reagents) assays are free.
Be sure you completely understand the lab report format: ask
questions now!